Hi Dhoouha.

You need to isolate cells in complete suspension and keep them in single cells all the time during specially FACS because the primary kidney cells would stick together to show larger particle or clumps during sorting process. Please read my two comments in the link. https://www.researchgate.net/post/FACS_single_cell_analysis-debris_dead_cells_or_artifact

Use Sigma lysis buffer to get rid of RBC. Have a look at similar topic discussions.

https://www.researchgate.net/post/What_lysis_protocols_can_I_use_to_efficiently_eliminate_red_blood_cell_from_whole_blood

https://www.researchgate.net/post/What_is_the_best_RBCs_lysis_buffer_could_be_used_for_the_isolation_of_circulating_tumour_cells_CTCs

Regards.

Hi Saang!

Thank you for your answer. That's what I am trying to do but still I find debris and dead cells. I am using adult kidney tissue. I tried 0.1% Triton X-100 with 50% methanol;

http://www.abcam.com/protocols/red-blood-cell-lysis-protocol-1

and it didn't work. Thank you for the recommendation. Kidney tissue are hard to prepare for FACS analysis without dead cells and debris I think.

Hi Dhoouha.

Make small pieces of kidney by chopping, followed by collagenase type 1 in Hank's solution for 1h at incubator, wash by gentle changes of fresh cold Hank's sol several times. It would be more effective to removing cell debris and dead cells by a trick of gentle whirling washes in a 100mm dish with enough sol. Gentle whirling on the bench 3~5 times. Lager pieces of tissues will be collected in the center of the dish, while cell debris and dead cells will disperse periphery of the dish. Using a Pasteur pipette or plastic dispenser, suck the periphery fluid and debris, and discard under a stereomicroscope stage underlight. Repeat this to clear the tissue pieces. Transfer into trypsin-EDTA in Erlenmeyer flask on gentle stirring for 10 min at RT. Save the clean suspension leaving the tissues-associated with ECM. immediately transfer the cell suspension into a equal vol of cold FBS on ice to stop the digestion. Spin, collect cells. Upside-down the 50ml tube containing the cells. and stand-up, followed by immediate taking the upper part of the cell suspension using the plastic dispenser to exclude cell clumps and undissociated tissues. Transfer the part of the save cell suspension into 100mm dish containing Hank's. Repeat the above gentle whirling procedure again to remove the cell debris and dead cells, several washes like this.

Thus, the procedure is not positive selection for healthy cells, but negative selection for dead cells except for the elimination procedure of cell clumps and tissues. Use 2% PFA for 20 min followed by 0.1% Trton X-100 for 30min to extract cells without using MeOH. MeOH would make your extracted cells cells brisk, leading debris during procedure of antibody staining. I would use 2mgBSA/ml after fixation before adding Triton X-100.(Just read it through, and you can make your own protocol defined.) Do not follow the instruction provided by the company mechanistically. jump into the tube with the cells.

Best wishes.

Hi Saang

Thank you so much for your suggestions.

Did you try this with adult mice kidney?because I know you work with embryo. are you sing the Erlenmeyer for the small volume of mouse kidney cell suspension?

I know someone in a neighbor lab who failed to get good quality single cells from kidney for cells sorting using the same lysis buffer you mentioned. My lysis buffer is less harsh than the one you suggested but the problem is that it includes enzymes that could be inhibited by FBS so I cannot add it. I also cannot fix my cells by PFA because I might want to collect them after sorting for another experiment. I got so far decent healthy alive cells quality and good cells number but still a lot of debris there when using the kidney cell enzymes based lysis buffer alone but I lost a lot of cells when using RBC buffer (ammonium chloride/ potassium chloride with EDTA) followed by my kidney cell lysis buffer... I know I can't avoid using the RBC lysis buffer but am not sure if its a good idea to keep both lysis buffer in my protocol..